Intestinal barrier integrity

ABSTRACT

The invention concerns a method for stimulating barrier integrity in a mammal by administering to a mammal a composition comprising: eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ARA), and at least two distinct oligosaccharides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/739,850, filed Jun. 15, 2015, which is a Continuation of U.S.application Ser. No. 13/594,147, filed Aug. 24, 2012, which is aContinuation of U.S. application Ser. No. 11/571,122, filed Sep. 6,2007, which is a National Stage entry of International Application No.PCT/NL2004/000444, filed Jun. 22, 2004, the entire contents of which arehereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a method for improving intestinalbarrier integrity and a composition suitable for use in such method.

BACKGROUND OF THE INVENTION

The gastrointestinal epithelium normally functions as a selectivebarrier permitting the absorption of nutrients, electrolytes and waterand preventing the exposure to dietary and microbial antigens, includingfood allergens. The gastrointestinal epithelium limits the passage ofantigens to the systemic circulation, that may be causing inflammatoryreactions, e.g. allergic reactions. As the incidence of allergy,particularly food allergy, is increasing, many research groups searchfor (preventive) cures for these ailments.

EP1272058 describes a composition containing indigestibleoligosaccharides for improving tight junction to reduce intestinalpermeability and reducing allergic reaction. The composition maycomprise LC-PUFA's (long chain-polyunsaturated fatty acids).

EP 745001 describes a combination of indigestible oligosaccharides andn-3 and n-6 fatty acids for treatment ulcerative colitis.

Usami et al (Clinical Nutrition 2001, 20(4): 351-359) describe theeffect of eicosapentaenoic acid (EPA) on tight junction permeability inintestinal monolayer cells. In their hands, EPA was found to increasepermeability, indicating that EPA is unsuitable to improve intestinalbarrier integrity.

The prior art formulations are not optimally suited for improvingbarrier integrity.

SUMMARY OF THE INVENTION

The present invention provides a combination of selected long chainpolyunsaturated fatty acids (LC-PUFA's) and selected oligosaccharides.The present combination of LC-PUFA's and oligosaccharides effectivelyimproves barrier integrity, by synergistically improving intestinalpermeability and mucus production, and is particularly suitable forimproving barrier integrity in human infants.

It was surprisingly found that selected LC-PUFA's effectively reduceepithelial paracellular permeability. In contrast to what Usami et al(Clinical Nutrition 2001, 20(4): 351-359) have reported, the presentinventors found that C18 and C20 polyunsaturated fatty acids,particularly eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) andarachidonic acid (ARA), are capable of effectively reducing intestinaltight junction permeability.

In addition to the LC-PUFAs, the present composition containsoligosaccharides. The selected oligosaccharides improve the barrierintegrity by stimulating the production of the mucus, which results inan increased mucus layer thickness. It is believed this effect is causedby the effects of the distinct oligosaccharides on the short chain fattyacid (SCFA) production. Hence, when enterally administered to a mammal,the present combination of LC-PUFA and indigestible oligosaccharidessynergistically improve harrier integrity and/or synergistically reduceintestinal permeability by simultaneous reduction of tight junctionpermeability and stimulation of mucus production.

In a further aspect, the present composition improves the quality of theintestinal mucus layer. The mucus layer comprises mucins. Mucins arehigh molecular mass glycoproteins that are synthesized and secreted bygoblet cells. They form a gel-like layer on the mucosal surface, therebyimproving barrier integrity. The mucus layer comprises different typesof mucins, e.g. acid, neutral and sulphonated mucins. An increasedheterogeneity of the mucus layer is believed to improve barrierfunctionality.

The present composition preferably comprises at least two differentoligosaccharides, which influence the mucosal architecture andadvantageously influence mucin heterogeneity in the mucus layer, eitherdirectly or by changing the intestinal flora. Each different selectedoligosaccharide is believed to have a different effect on mucus quantityand quality. Moreover, the two distinct oligosaccharides are also ableto stimulate quality of mucus as reflected by the degree of sulphationthrough their synergistic stimulation of SCFA production. It wassurprisingly found by the present inventors that a mixture of twodifferent oligosaccharides according to the present inventionsynergistically stimulates acetate production. It was also found by thepresent inventors mucus production is dependent on acetate production.

The present composition is preferably further improved by providing bothlong- and short-chain oligosaccharides. The supply of different chainlengths results in stimulation of mucus production in different parts ofthe ileum and colon. The short chain oligosaccharides (typically with adegree of polymerisation (DP) of 2,3,4 or 5) stimulate mucin productionin the proximal colon and/or distal ileum, while the oligosaccharideswith longer chain lengths (preferably with a degree of polymerisation(DP) of more than 5 up to 60) are believed to stimulate mucin productionin the more distal parts of the colon.

Even further improvements can be achieved by providing the at least twodifferent oligosaccharides both as short-chain and long-chainoligosaccharides. These preferred embodiments all contribute to furtherimproved barrier integrity throughout the ileum and/or colon.

Furthermore, it was surprisingly found that EPA, DHA and ARA were ableto reduce the harmful effects of interleukin 4 (IL-4) on intestinalpermeability. IL-4 is a cytokine which is secreted in increased amountsby mucosal T-cells in certain patients and induces intestinalpermeability. Hence the present invention also provides for a method forthe treatment and/or prevention of diseases wherein intestinal IL-4concentration is increased, such as allergy, particularly atopicdermatitis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the time and dose dependent effects of palmiticacid and DHA (FIG. 1A) and GLA and AA (FIG. 1B) on basal barrierintegrity (TER).

FIGS. 2A and 2B give the time and dose dependent protective effects ofpalmitic acid and DHA (FIG. 2A) and GLA and AA (FIG. 2B) on IL-4mediated barrier destruction (Flux).

FIGS. 3A and 3B depict the absolute (FIG. 3A) and relative SCFA profile(FIG. 3B) resulting from fermenting the different oligosaccharides.

FIG. 4 shows the differential effects of SCFA (acetate, proprionate,butyrate) on MUC-2 expression in intestinal epithelial cells (MC T84)and epithelial-mesenchymal cell co-cultures (CC T84).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a nutritional composition comprising:

-   a) EPA, DHA and ARA, wherein the content of long chain    polyunsaturated fatty acid with 20 and 22 carbon atoms does not    exceed 15 wt. % of the total fat content; and-   b) at least two distinct oligosaccharides, wherein the two distinct    oligosaccharides have a homology in monose units below 90%.

This composition can be advantageously used in a method for stimulatingintestinal barrier integrity, said method comprising administering to amammal said composition.

Polyunsaturated Fatty Acids

The present inventors surprisingly found that eicosapentaenoic acid(EPA, n-3), docosahexaenoic acid (DHA, n-3) and arachidonic acid (ARA,n-6) effectively reduce intestinal tight junction permeability. Hencethe present composition, which is particularly suitable for improvingintestinal barrier integrity, comprises EPA, DHA and ARA.

The present inventors found that lower concentration of LC-PUFA's, wereeffective in reducing tight junction permeability (see Examples vs.Usami et al). Hence, the content of LC-PUFA with 20 and 22 carbon atomsin the present composition, preferably does not exceed 15 wt. % of thetotal fat content, preferably does not exceed 10 wt. %, even morepreferably does not exceed 5 wt. % of the total fat content. Preferablythe present composition comprises at least 0.1 wt. %, preferably atleast 0.25 wt, more preferably at least 0.5 wt. %, even more preferablyat least 0.75 wt. % LC-PUFA with 20 and 22 carbon atoms of the total fatcontent. For the same reason, the EPA content preferably does not exceed5 wt. % of the total fat, more preferably does not exceed 1 wt. %, butis preferably at least 0.05 wt %, more preferably at least 0.1 wt. % ofthe total fat. The DHA content preferably does not exceed 5 wt. %, morepreferably does not exceed I wt. %, but is at least 0.1 wt % of thetotal fat. As ARA was found to be particularly effective in reducingtight junction permeability, the present composition comprisesrelatively high amounts, preferably at least 0.1 wt. %, even morepreferably at least 0.25 wt. %, most preferably at least 0.5 wt. % ofthe total fat. The ARA content preferably does not exceed 5 wt. %, morepreferably does not exceed 1 wt. % of the total fat. In the present ARAcontaining enteral composition, EPA and DHA are advantageously added tobalance the action of ARA, e.g. reduce the potential proinflammatoryaction of ARA metabolites. Excess metabolites from ARA may causeinflammation. Hence, the present composition preferably comprises ARA,EPA and DHA, wherein the weight ratio ARA/DHA preferably is above 0.25,preferably above 0.5, even more preferably above 1. The ratio ispreferably below 25. The weight ratio ARA/EPA is preferably between 1and 100, more preferably between 5 and 20.

The present composition preferably comprises between 5 and 75 wt. %polyunsaturated fatty acids based on total fat, preferably between 10and 50 wt. %.

If the present composition is used as an infant formula (e.g. a methodfor feeding an infant, said method comprising administering the presentcomposition to an infant), the content of LC-PUFA, particularly theLC-PUFA with 20 and 22 carbon atoms, preferably does not exceed 3 wt. %of the total fat content as it is desirable to mimic human milk asclosely as possible. For the same reason, the omega-3 LC-PUFA contentpreferably does not exceed 1 wt. % of the total fat content; the omega-6LC-PUFA content preferably does not exceed 2 wt. % of the total fatcontent; the ARA (omega-6) content is preferably below 1 wt. % of thetotal fat content; and/or the weight ratio EPA/DHA is preferably 1 orlower, more preferably below 0.5.

The LC-PUFA with 20 and 22 carbon atoms may be provided as free fattyacids, in triglyceride form, in phospholipid form, or as a mixture ofone of more of the above. The present composition preferably comprisesat least one of ARA and DHA in phospholipid form.

The present nutritional composition preferably also provides omega-9(n-9) fatty acid (preferably oleic acid, 18:1), to provide sufficientnutrition. Preferably the present composition provides at least 15 wt. %n-9 fatty acid based on the weight of the total fatty acids, morepreferably at least 25 wt %. The content of n-9 fatty acids ispreferably below 80 wt. %.

Oligosaccharides

Suitable oligosaccharides according to the invention are saccharideswhich have a degree of polymerisation (DP) of at least 2 monose units,which are not or only partially digested in the intestine by the actionof acids or digestive enzymes present in the human upper digestive tract(small intestine and stomach), but which are fermentable by the humanintestinal flora. The term monose units refers to units having a closedring structure, preferably hexose, e.g. the pyranose or furanose forms.The degree of polymerisation of the oligosaccharide is typically below60 monose units, preferably below 40, even more preferably below 20.

The present composition comprises at least two differentoligosaccharides, wherein the oligosaccharides have a homology in monoseunits below about 90%, preferably below 50%, even more preferably below25%, even more preferably below 5%. The term “homology” as used in thepresent invention is the cumulative of the percentage of same monoseunits in the different oligosaccharides. For example, oligosaccharide 1(OL1) has the structure fruc-fruct-glu-gal, and thus comprises 50% fruc,25% gal and 25% glu. Oligosaccharide 2 (OL2) has the structurefruc-fruc-glu, and thus comprises 66% fruc, 33% glu. The differentoligosaccharides thus have a homology of 75% (50% fruc+25% glu).

In a preferred embodiment, the present composition comprisesgalactooligosaccharides and at least one selected from the groupconsisting of fructooligosaccharides and inulin.

Each of the present oligosaccharides preferably comprises at least 66%,more preferably at least 90% monose units selected from the groupconsisting of mannose, arabinose, fructose, fucose, rhamnose, galactose,β-D-galactopyranose, ribose, glucose, xylose, uronic acid andderivatives thereof, calculated on the total number of monose unitscontained therein.

According to a further embodiment at least one of the oligosaccharidesof the present composition is selected from the group consisting offructans, fructooligosaccharides, indigestible dextrinsgalactooligosaccharides (including transgalactooligosaccharides),xylooligosaccharides, arabinooligosaccharides, glucooligosaccharides,mannooligosaccharides, fucooligosaccharides, acidic oligosaccharides(see below, e.g. uronic acid oligosaccharides such as pectinhydrolysate) and mixtures thereof. Preferably the present compositioncomprises at least one, preferably at least two, of the oligosaccharidesselected from the group consisting of fructooligosaccharides or inulin,galactooligosaccharides and pectin hydrolysate.

For good mucus quantity and quality, the present composition preferablycomprises at least one oligosaccharide, which comprises at least 66%galatose or fructose as a monose unit. In a preferred embodiment thecomposition comprises at least one oligosaccharide which comprises atleast 66% galatose as a monose unit and at least one oligosaccharidewhich comprises at least 66% fructose as a monose unit. In aparticularly preferred embodiment, the present composition comprisesgalactooligosaccharide and an oligosaccharide selected from the groupconsisting of fructooligosaccharides and inulin. Fructooligosaccharidesstimulate sulfomucin production in the distal colon of humanflora-associated rats (Kleessen et al, (2003) Brit J Nutr 89:597-606)and galactooligosaccharides stimulate the acid mucin production (Meslinet al, Brit. J. Nutr (1993), 69: 903-912)).

For further improvement of mucus layer thickness over the whole area ofthe colon, at least 10 wt. % of the oligosaccharides in the presentcomposition has a DP of 2 to 5 (i.e. 2, 3, 4 and/or 5) and at least 5wt. % has a DP of 10 to 60. Preferably at least 50 wt. %, morepreferably at least 75 wt. % of the oligosaccharides have a DP of 2 to 9(i.e. 2, 3, 4, 5, 6, 7, 8, and/or 9), because these are believed to workthroughout the ileum and proximal and middle parts of the colon andbecause the weight percentage of oligosaccharides that needs to beincorporated in the composition to achieve the desired effect isreduced.

Preferably the weight ratios:

-   a. (oligosaccharides with DP 2 to 5):(oligosaccharides with DP 6,7,8    and/or 9)>1; and-   b. (oligosaccharides with DP 10 to 60):(oligosaccharides with DP    6,7,8 and/or 9)>1 are both above 1.

Preferably both weight ratios are above 2, even more preferably above 5.

For even further improvement of mucus layer thickness and quality overthe whole area of the colon, preferably each of the at least twodifferent oligosaccharides are provided in different chain lengths,preferably at least 10 wt. % of each oligosaccharide based on the totalweight of the respective oligosaccharide has a DP of 2 to 5 (i.e. 2, 3,4 and/or 5) and at least 5 wt. % has a DP between 10 and 60. Preferablyat least 50 wt. %, more preferably at least 75 wt. % of theoligosaccharide based on the total weight of that oligosaccharides has aDP between 2 and 10, because these are believed to work throughout inthe ileum and proximal and middle parts of the colon.

Acidic Oligosaccharides

To further improve barrier integrity, the present composition preferablyincludes acidic oligosaccharides with a DP between 2 and 60. The termacid oligosaccharide refers to oligosaccharides comprising at least oneacidic group selected from the group consisting of N-acetylneuraminicacid, N-glycoloylneuraminic acid, free or esterified carboxylic acid,sulfuric acid group and phosphoric acid group. The acidicoligosaccharide preferably comprises uronic acid units (i.e. uronic acidpolymer), more preferably galacturonic acid units. The acidoligosaccharide may be a homogeneous or heterogeneous carbohydrate.Suitable examples are hydrolysates of pectin and/or alginate. In theintestinal tract, the uronic acid polymers are hydrolysed to uronic acidmonomers, which stimulate production of intestinal acetate, which inturn stimulates intestinal mucus secretion (Barcelo et al., Gut 2000;46:218-224).

Preferably the acid oligosaccharide has the structure I below, whereinthe terminal hexose (left) preferably comprises a double bond. Thehexose units other than the terminal hexose unit(s) are preferablyuronic acid units, even more preferably galacturonic acid units. Thecarboxylic acid groups on these units may be free or (partly)esterified, and preferably at least 10% is methylated (see below).

wherein:R is preferably selected from the group consisting of hydrogen, hydroxyor acid group, preferably hydroxy; andat least one selected from the group consisting of R₂, R₃, R₄ and R₅represents N-acetylneuraminic acid, N-glycoloylneuraminic acid, free oresterified carboxylic acid, sulfuric acid group and phosphoric acidgroup, and the remaining of R₂, R₃, R₄ and R₅ representing hydroxyand/or hydrogen. Preferably one selected from the group consisting ofR₂, R₃, R₄ and R₅ represents N-acetylneuraminic acid,N-glycoloylneuraminic acid, free or esterified carboxylic acid, sulfuricacid group or phosphoric acid group, and the remaining represent hydroxyand/or hydrogen. Even more preferably one selected from the groupconsisting of R₂, R₃, R₄ and R₅ represents free or esterified carboxylicacid and the remaining of R₂, R₃, R₄ and R₅ representing hydroxy and/orhydrogen; andn is an integer and refers to a number of hexose units (see also Degreeof Polymerisation, below), which may be any hexose unit. Suitably n isan integer between 1-5000. Preferably the hexose unit(s) is a uronicacid unit.

Most preferably R₁, R₂ and R₃ represent hydroxy, R₄ represent hydrogen,R₅ represents carboxylic acid, n is any number between 1 and 250,preferably between 1 and 10 and the hexose unit is galacturonic acid.

The detection, measurement and analyses of the preferred acidoligosaccharides as used in the present method are given in applicantsearlier patent application relating to acid oligosaccharides, i.e. WO0/160378.

For stimulation improvement of mucus layer thickness over the whole areaof the colon, the present composition preferably comprises at least 10wt. % acid oligosaccharides with a DP of 2 to 5 (i.e. 2, 3, 4 and/or 5)and at least 5 wt. % acid oligosaccharides with a DP between 10 and 60,said wt. % being based on the total weight of the oligosaccharides.

The acid oligosaccharides used in the invention are preferably preparedfrom pectin, pectate, alginate, chondroitine, hyaluronic acids,heparine, heparane, bacterial carbohydrates, sialoglycans, fucoidan,fucooligosaccharides or carrageenan, more preferably from pectin and/oralginate.

Content of Oligosaccharide

When in ready-to-feed liquid form, the present composition preferablycomprises 0.1 to 100 grams indigestible oligosaccharide per liter, morepreferably between 0.5 and 50 grams per liter even more preferablybetween 1 and 25 grams per liter. A too high content of oligosaccharidesmay cause discomfort due to excessive fermentation, while a very lowcontent may result in an insufficient mucus layer.

The weight ratio of the at least two different oligosaccharides ispreferably between 1 and 10, more preferably between 1 and 5. Theseweight ratios stimulate mucin production of different types at differentsites in the intestine optimally.

The oligosaccharide is preferably included in the present compositionaccording to the invention in an amount exceeding 0.1 wt. %, preferablyexceeding 0.2 wt. %, more preferably exceeding 0.5 wt. % and even morepreferably exceeding 1 wt. % based on the total dry weight of thecomposition. The present composition preferably has an oligosaccharidecontent below 20-wt. %, more preferably below 10-wt. % even morepreferably below 5-wt. %.

Addition of nucleotides and/or nucleosides to the present compositionfurther improves gut mucosal barrier function, particularly as itinhibits and/or or reduces the incidence of bacterial translocation anddecreases intestinal injury. Hence, the present composition preferablyalso comprises between 1 and 500 mg nucleosides and/or nucleotides per100 gram of the dry formula, even more preferably between 5 and 100 mg.

Application

The present composition can be advantageously used in a method forimproving barrier integrity in mammals, particularly humans. The presentcomposition can also be advantageously used in a method for thetreatment or prevention of diseases associated with reduced barrierintegrity, said method comprising administering to a mammal the presentcomposition. The present composition is preferably administered orally.

For the ill and infants, the present composition is preferably combinedwith complete nutrition, including protein, carbohydrate and fat. Thepresent composition is advantageously administered to infants with theage between 0 and 2 years. The composition may be administered topatients which suffer from an impaired barrier integrity and healthypatients. The present composition is advantageously used in a method forproviding the nutritional requirements of a premature infant (an infantborn before 37 weeks gestation).

The present composition can also be advantageously used in a method fortreatment and/or prevention of intestinal damage by administering thepresent composition to the patient prior to or after a medicaltreatment, which may cause intestinal damage. Such medical treatment mayfor example be surgery or enteral medicine treatment (e.g. antibiotic,analgesic, NSAID, chemotherapeutic agents etc).

The present composition can also be advantageously used to treat orprevent diseases wherein intestinal barrier disruption is underlying thedevelopment of the course of the disease, e.g. in a method for thetreatment or prevention of chronic inflammatory diseases, particularlyinflammatory bowel disease (IBD), irritable bowel syndrome (IBS), celiacdisease, pancreatitis, hepatitis, arthritis or diabetes. Furthermore,the invention can be used in a method for providing nutrition topatients which have undergone or are undergoing abdominal surgery andpatients that experience postoperative dysfunction of the gut and/ormalnourished patients.

In a further embodiment of the invention the present composition isadvantageously administered to patients suffering from acquired immunedeficiency syndrome (AIDS) and/or patients which are infected with thehuman immunodeficiency virus (HIV), e.g. in a method for the treatmentof AIDS and/or HIV infection. Said method comprises the oraladministration of the present composition, preferably combined withnutrients selected from the group consisting of carbohydrate, proteinand fat.

Furthermore, the invention can also be used to treat or preventcomplications resulting from reduced barrier integrity, particularly ina method for the treatment and/or prevention of diarrhea, particularlyinfant diarrhea. Due to the reduced incidence in infant diarrhea, thepresent composition can also be advantageously used to reduce diaperrash.

Administering the present composition reduces passage of dietary andmicrobial antigens, particularly food allergens, from the intestinallumen into the mucosa] or systemic circulation, and hence can beadvantageously used in a method for the treatment or prevention ofallergy and/or allergic reaction, particularly in a method for thetreatment or prevention of food allergy, e.g. allergic reactionresulting from the ingestion of foodstuff.

It was also found by the present inventors that EPA, DHA and/or ARA arecapable of reducing the effects of IL-4 on intestinal permeability.Hence, one aspect of the present invention provides for a method for thetreatment and/or prevention of diseases wherein intestinal IL-4concentration is increased (e.g. allergic diseases), said methodcomprising administering an LC-PUFA preferably selected from the groupconsisting of EPA, DHA and ARA, preferably combined with the presentselected oligosaccharides. Hence, the present composition can also beadvantageously used in a method for the treatment of atopic dermatitis.

Since the barrier function of newborns has not been fully developed, thepresent composition can be advantageously administered to young infants,i.e. infants with the age between 0 and 6 months. The composition may beadministered to the infant in the form of an infant formula withouthuman milk or admixed with human milk. Hence the present invention alsoprovides for a formula feed comprising human milk and the presentcomposition. The compositions including human milk and the presentcomposition are particularly suitable for feeding premature infants.

The present composition is preferably provided as a packaged powder orpackaged ready-to-feed formula. To prevent spoilage of the product,packaging size of ready-to-feed formula preferably does not exceed oneserving, e.g. preferably does not exceed 500 ml; and packaging size ofthe present composition in powder form preferably does not exceed 250servings. Suitable packaging sizes for the powder are 2000 grams orless, preferably per 1000 grams or less.

The packaged products provided with labels that explicitly or implicitlydirect the consumer towards the use of said product in accordance withone or more of the above or below purposes, are encompassed by thepresent invention. Such labels may for example make reference to thepresent method for preventing allergic reaction to food allergens byincluding wording like “reduced food sensitivity”, “improving intestinaltolerability”, “improved food tolerance” or similar wording. Similarly,reference to the present method for treating and/or preventing allergymay be made by incorporating terminology equivalent to “improvedresistance” or “reduced sensitivity”.

Formula's

It was found that the present composition can be advantageously appliedin food, such as baby food and clinical food. Such food preferablycomprises lipid, protein and carbohydrate and is preferably administeredin liquid form. The term “liquid food” as used in the present inventionincludes dry food (e.g. powders) which are accompanied with instructionsas to admix said dry food mixture with a suitable liquid (e.g. water).

Hence, the present invention also relates to a nutritional compositionwhich preferably comprises between 5 and 50 en % lipid, between 5 and 50en % protein, between 15 and 90 en % carbohydrate and the presentcombination of oligosaccharides and LC-PUFA's. Preferably the presentnutritional composition preferably contains between 10 and 30 en %lipid, between 7.5 and 40 en % protein and between 25 and 75 en %carbohydrate (en % is short for energy percentage and represents therelative amount each constituent contributes to the total caloric valueof the preparation).

Preferably a combination of vegetable lipids and at least one oilselected from the group consisting of fish oil and omega-3 vegetable,algae or bacterial oil is used.

The proteins used in the nutritional preparation are preferably selectedfrom the group of non-human animal proteins (such as milk proteins, meatproteins and egg proteins), vegetable proteins (such as soy protein,wheat protein, rice protein, and pea protein), free amino acids andmixtures thereof. Cow milk derived nitrogen source, particularly cowmilk protein proteins such as casein and whey proteins are particularlypreferred.

A source of digestible carbohydrate may be added to the nutritionalformula. It preferably provides about 40% to about 80% of the energy ofthe nutritional composition. Any suitable (source of) carbohydrate maybe used, for example sucrose, lactose, glucose, fructose, corn syrupsolids, and maltodextrins, and mixtures thereof.

The present composition is preferably used as an infant formula andpreferably contains 7.5 to 12.5 energy % protein; 40 to 55 energy %carbohydrates; and 35 to 50 energy % fat. As the present composition issuitably used to reduce the allergic reaction in an infant, the proteinof the infant formula is preferably selected from the group consistingof hydrolyzed milk protein (e.g. hydrolyzed casein or hydrolyzed wheyprotein), vegetable protein and/or amino acids. The use of theseproteins further reduced the allergic reactions of the infant.

Stool irregularities (e.g. hard stools, insufficient stool volume,diarrhea) is a major problem in many babies and ill subjects thatreceive liquid foods. It was found that stool problems may be reduced byadministering the present oligosaccharides in liquid food which have anosmolality between 50 and 500 mOsm/kg, more preferably between 100 and400 mOsm/kg.

In view of the above, it is also important that the liquid food does nothave an excessive caloric density, however still provides sufficientcalories to feed the subject. Hence, the liquid food preferably has acaloric density between 0.1 and 2.5 kcal/ml, even more preferably acaloric density of between 0.5 and 1.5 kcal/ml, most preferably between0.6 and 0.8 kcal/mi.

EXAMPLES Example 1: Effect of LC-PUFA on Barrier Integrity

Monolayers (MC) of intestinal epithelial cell lines T84 (American TypeCulture Collection (ATTC), Manassas, USA) were cultured on transwellfilters (Corning, Costar BV, The Netherlands) allowing both mucosal andserosal sampling and stimulation of human intestinal epithelial cells.Two weeks post confluency the monolayers were incubated in the luminalcompartment with polyunsaturated fatty acids ARA (arachidonic acid;5,8,11,14-eicosatetraenoic acid), DHA (cis-4,7,10,13,16,19docosahexaenoic acid), EPA (eicosapentaenoic acid) or control palmitic(C 16:0) acid (Palm) (Sigma, St. Louis, USA). The latter procedure waschosen to mimic the in vivo administration route of the dietarycompounds. Cells were incubated with ARA, DHA, EPA, or palmitic acid for0, 24, 48 and 72 hr at different concentrations (10 μM and 100 μM).Experiments were performed to evaluate basal barrier integrity. Theepithelial barrier function was determined by measuring thetransepithelial resistance (TER, Ω·cm²) was measured by epithelialvolt-ohm meter (EVOM; World Precision Instruments, Germany) andpermeability for 4 kD FITC dextran (paracellular permeability marker,Sigma, USA). Resistance (Epithelial permeability for 4 kDa FITC-dextranwas determined as follows. Prior to dextran fluxes the medium wasrefreshed with culture medium without phenol red for one hour followedby addition of 5 μl (stock 100 mg/ml) 4 kDa FITC-dextran to the lumenalcompartment. After 30 min incubation 100 μl sample was collected fromthe serosal compartment and the fluorescent signal measured atexcitation wavelength 485 nm and emission 520 nm (FLUOstar Galaxy®, BMGLabtechnologies, USA). FITC-dextran fluxes were calculated as pmolFITC-dextran/cm²/h. Statistical analyses were performed using the ANOVA(SPSS version 10).

Results of the effect of fatty acids (100 μM) on spontaneous barrierintegrity after 72 hr incubation are given in Table 1. Table 1 showsthat the LC-PUFA's ARA, EPA and DHA reduce the molecular flux andimprove epithelial resistance. In contrast the control experiments showthat palmitic acid has the opposite effects, i.e. compromises barrierintegrity. These results are indicative for the advantageous use of EPA,DHA and ARA, and in particularly ARA in the composition according to thepresent invention and for use in a method according to the presentinvention, e.g. in a method for improving barrier integrity. Theseresult further support the synergistic effects of the presentcombination of fatty acids and indigestible oligosaccharides.

FIG. 1 shows the time and dose (10 μM and 100 μM) dependent effects ofvarious fatty acids (palmitic acid, DHA, GLA, and AA) on basal barrierintegrity (TER). FIG. 1 shows that the LC-PUFA's AA, DHA, and GLA,improve the epithelial barrier integrity as reflected by increasedresistance (TER). These results are indicative for the advantageous useof EPA, DHA, GLA and ARA, in particularly ARA, in the compositionaccording to the present invention and for use in a method according tothe present invention, i.e. in a method for improving barrier integrity.These results further support the synergistic effects of the presentcombination of fatty acids and indigestible oligosaccharides.

TABLE 1 Ingredient (LC-PUFA) Flux Resistance (TER) Control 79 1090Palmitic acid 161 831 DHA 72 1574 ARA 28 1816 EPA 65 1493

Example 2: Effect of LC-PUFA on IL-4 Mediated Barrier Disruption

Monolayers (MC) of intestinal epithelial cell lines T84 (ATCC, USA) werecultured on transwell filters (Corning, Costar BV, The Netherlands)allowing both mucosal and serosal sampling and stimulation of humanintestinal epithelial cells. Two weeks post confluency the monolayerswere incubated in the presence of IL-4 (2 ng/ml, serosal compartment,Sigma, USA) with or without polyunsaturated fatty acids ARA, DHA, GLA,EPA, or control palmitic acid (10 μM or 100 μM, mucosal compartment,Sigma, St. Louis, USA). Cells were pre-incubated with ARA, DHA, EPA, orpalmitic acid for 48 hr prior to the IL-4 incubation. The co-incubationof PUFA's and palmetic acid with IL-4 was continued for another 48 hr;while culture medium and additives were changed every 24 hr. Theepithelial barrier function was determined by measuring thetransepithelial resistance (TER) and permeability as described inexample 1. Statistical evaluation was performed as described in example1.

Results of the effect of ARA, DHA, EPA and palmitic acid (100 μM) onIL-4 mediated barrier disruption are given in Table 2. Table 2 showsthat the LC-PUFA's ARA, DHA and EPA inhibit the increased flux caused byIL-4. In contrast palmetic acid had a detrimental effect and decreasedbarrier disruption compared to control. These results are indicative forthe advantageous use of ARA, DHA, and EPA in clinical and infantnutrition formulations to prevent or reduce IL-4 mediated barrierdisruption, e.g. as occurs in food or cows milk allergy. These resultfurther support the synergistic effects of the present combination offatty acids and indigestible oligosaccharides.

FIG. 2 gives the time and dose (10 μM and 100 μM) dependent protectiveeffects of various FA's (palmitic acid, DHA, GLA, and AA) on IL-4mediated barrier destruction (Flux). FIG. 2 shows that ARA, DHA and GLAprotect against IL-4 mediated barrier disruption as reflected bydecreased 4 kD dextran flux. These results are indicative for theadvantageous use of ARA, DHA and GLA in clinical and infant nutritionformulations to prevent or reduce IL-4 mediated barrier disruption, e.g.as occurs in food or cows milk allergy. These result further support thesynergistic effects of the present combination of fatty acids andindigestible oligosaccharides.

TABLE 2 Ingredient (LC-PUFA) IL-4 Flux IL-4 TER Control 582 374 Palmiticacid 777 321 DHA 271 547 ARA 218 636 EPA 228 539

Example 3: Effect of Oligosaccharides on Acetate Production

Micro-organisms were obtained from fresh faeces from bottle fed babies.Fresh faecal material from babies ranging 1 to 4 month of age was pooledand put into preservative medium within 2 h. As substrate eitherprebiotics (TOS; TOS/inulin (HP) mixture in a 9/1 (w/w) ratio; inulin;oligofructose(OS)/inulin mixture in a 1/1 (w/w) ratio, or none (blanc)were used. The transgalactooligosaccharides (TOS) were obtained fromVivinal GOS, Borculo Domo Ingredients, Zwolle, The Netherlands andcomprises as indigestible oligosaccharides: 33 wt. % disaccharides, 39wt. % trisaccharides, 18 wt. % tetrasaccharides, 7 wt. %pentasaccharides and 3 wt. % hexa-, hepta-en octasaccharides. The inulin(HP) Orafti active food ingredients, Tienen, Belgium, i.e. RaftilineHP®, with an average DP of 23. Media: McBain & MacFarlane medium:buffered peptone water 3.0 g/1, yeast extract 2.5 g/l. mucin (brushborders) 0.8 g/l, tryptone 3.0 g/1, L-Cysteine-HCl 0.4 g/l, bile salts0.05 g/l, K2HPO4.3H2O 2.6 g/l, NaHCO₃0.2 g/l, NaCl 4.5 g/l, MgSO4.7H2O0.5 g/l, CaCl2 0.228 g/l, FeSO4.7H2O 0.005 g/l. Fill 500 ml Scottbottles with the medium and sterilized 15 minutes at 121° C. Bufferedmedium: K2HPO4.3H2) 2.6 g/l, NaHCO₃0.2 g/l, NaCl 4.5 g/l, MgSO4.7H2O,0.5 g/l, CaCl2 0.228 g/l, FeSO4.7H2O 0.005 g/l. Adjust to pH 6.3±0.1with K2HPO4 or NaHCO₃. Fill 500 ml Scott bottles with the medium andsterilized 15 minutes at 121° C.

Preservative medium: Buffered peptone 20.0 g/l, L-Cysteine-HCl 0.5 g/l,Sodium thioglycollate 0.5 g/l, resazurine tablet 1 per litre, adjust topH 6.7±0.1 with 1 M NaOH or HCl. Boiled in microwave. Serum bottles werefilled with 25 ml medium and sterilized for 15 minutes at 121° C.

Fresh faecal samples were mixed with preservative medium and stored forseveral hours at 4° C. The preserved solution of faeces was centrifugedat 13,000 rpm for 15 minutes, supernatant removed and faeces mixed withMcBain & Mac Farlane medium in a weight ratio of 1:5. Of this faecalsuspension 3 ml were combined with 85 mg glucose or prebiotic or with noaddition (blanc) in a bottle and mixed thoroughly. A t=0 sample waswithdrawn (0.5 ml). 2.5 ml of the resulting suspension is brought in adialysis tube in a 60 ml bottle filled with 60 ml of the bufferedmedium. The bottle was closed well and incubated at 37° C. Samples weretaken from the dialysis tube (0.2 ml) or dialysis buffer (1.0 ml) with ahypodermic syringe after 3, 24, and 48 hours and immediately put it onice to stop fermentation. The experiment was carried out using thefollowing samples:

-   1) 85 mg TOS-   2) 85 mg inulin-   3) 85 mg TOS/inulin in a ratio of 9/1 (w/w) and-   4) 85 mg OS/inulin in a ratio of 1/1 (w/w).

SCFA (acetate, propionate, butyrate) were quantitated using a Varian3800 gas chromatograph (GC) (Varian Inc., Walnut Creek, U.S.A.) equippedwith a flame ionisation detector. 0.5 μl of the sample was injected at80° C. in the column (Stabilwax, 15×0.53 mm, film thickness 1.00 μm,Restek Co., U.S.A.) using helium as a carrier gas (3.0 psi). Afterinjection of the sample, the oven was heated to 160° C. at a speed of16° C./min, followed by heating to 220° C. at a speed of 20° C./min andfinally maintained at 220° C. for 1.5 minutes. The temperature of theinjector and detector was 200° C. 2-ethylbytyric acid was used as aninternal standard.

FIG. 3 depicts the absolute (FIG. 3A) and relative SCFA profile (FIG.3B) resulting from fermenting the different oligosaccharides. FIG. 3Ashows that a mixture of two different oligosaccharides (TOS/Inulin),wherein the two distinct oligosaccharides have a homology in monoseunits below 90 and a different chain length results in a significantlyand synergistically increased amount of SCFA (particularly acetate) pergram fiber than single components. FIG. 3B shows that the addition of acombination of TOS/Inulin favored a higher proportion of the beneficialacetate (B). The acetate production in vivo translates to improved mucusproduction by goblet cells and a measure for intestinal mucus layerthickness (see example 4). These results are indicative for theadvantageous use of the present composition.

Example 4: Effects of SCFA on Mucus Production

Monolayers of intestinal epithelial T84 cells (ATCC, USA) cells werecultured in 24 or 96 wells tissue culture plates (Corning B.V.). T84were incubated with the short chain fatty acids acetate, proprionate andbutyrate (SCFA, Merck, USA) for 24 h in a concentration range of0.025-4.0 mM. Supernatants and/or cells were collected and MUC-2 (mucin)expression determined. A dotblot technique was used to determine MUC-2expression in the cell cultures, since mucins are extremely largeglycoproteins (over 500 kDa) which makes them difficult to handle inwestern blotting techniques. The method was validated using pre-immuneserum (T84 stained negative), CCD-18Co (ATCC, USA) negative controlcells and bovine serum albumin (BSA). Cell samples were collected inLaemmli (protein isolation buffer) and protein determination performedusing a microprotein assay (Biorad, USA) according to the manufacturersprotocol. Samples (0.3-0.7-1.0 μg/2 μl) were dotted on nitrocellulosemembranes (Schleicher & Schuell, Germany). Membranes were blocked inTBST/5% Protivar (Nutricia, The Netherlands) followed by 1 h incubationwith anti-MUC-2 antibody (kindly donated by Dr. Einerhand, ErasmusUniversity, Rotterdam, The Netherlands). After washing, blots wereincubated with goat anti-rabbit-HRP (Santacruz Biotechnology, USA) andfor substrate detection ECL (Roche Diagnostics, The Netherlands) wasused. Densitometry was performed using the Lumi-Imager (BoehringerMannheim B. V., The Netherlands) and the signal was expressed in lightunits (BLU). BLU's were also expressed relative to control incubations(% BLU). To compare the stimulatory effect of SCFA on MUC-2 expressionbasal MUC-2 expression levels were deducted.

FIG. 4 shows the differential effects of SCFA (acetate, proprionate,butyrate) on MUC-2 expression in intestinal epithelial cells (MC T84)and epithelial-mesenchymal cell co-cultures (CC T84). FIG. 2 also showsthat acetate is more potent in stimulating MUC-2 expression (mucusproduction) as compared to propionate and butyrate. Hence, the presentcombination of oligosaccharides (which was shown to increase acetateproduction (see example 3)) is particularly useful for stimulating mucusproduction and can be advantageously used in a method for stimulatingbarrier integrity.

Example 5: Infant Milk Formula I

Ingredients (per liter), energy 672 Kcal; Protein 15 g; Whey:Caseinratio 60:40; Fat 36 g; Carbohydrate 72 g; Vitamin A 750 RE; Mixednatural carotids 400 IU; Vitamin D 10.6 mcg; Vitamin F 7.4 mg; Vitamin K67.0 mcg; Vitamin B.sub. 1 (thiamin) 1000 mcg; Vitamin B.sub.2(riboflavin) 1500 mcg; Vitamin B.sub.6 (pyridoxine) 600 mcg; VitaminB.sub.12 (cyanacobalmine) 2.0 mcg; Niacin 9.0 mcg; Folic Acid 80 mcg;Pantothenic Acid 3000 mcg; Biotin 90 mcg; Vitamin C (ascorbic acid) 90mg; Choline 100 mg; Inositol 33 mg; Calcium 460 Mg; Phosphorous 333 Mg;Magnesium 64 Mg; Iron 8.0 Mg; Zinc 6.0 Mg; Manganese 50 mcg; Copper 560mcg; Iodine 100 mcg; Sodium 160 mg; Potassium 650 mg; Chloride 433 mgand Selenium 14 mcg; wherein the fat content provides includes 3 gramfish oil and 3 grams 40% arachidonic acid oil (DSM Food Specialties,Delft, Netherlands); further comprising 4 gramtransgalactooligosaccharides Elix'or™ (Borculo Domo Ingredients,Netherlands) and 4 gram Raftiline™ (Orafti Active Food Ingredients,Belgium).

The invention claimed is:
 1. A method for treating allergy, comprising administering to a subject in need thereof a nutritional composition comprising: (a) eicosapentaenoic acid (EPA) and arachidonic acid (ARA), wherein the content of long chain polyunsaturated fatty acid with 20 and 22 carbon atoms does not exceed 15 wt. % of the total fat content; and (b) at least two distinct oligosaccharides with a degree of polymerisation (DP) between 2 and 60 comprising transgalactooligosaccharide (TOS) and inulin.
 2. The method according to claim 1, wherein at least 10 wt. % of the oligosaccharides has a degree of polymerisation (DP) between 2 to 5 and at least 5 wt. % of the oligosaccharides has a DP between 10 and
 60. 3. The method according to claim 1, wherein the composition further comprises an acidic oligosaccharide with a DP between 2 and
 60. 4. The method according to claim 3, wherein the acidic oligosaccharide is an uronic acid polymer with a DP between 2 and
 6. 5. The method according to claim 1, wherein the composition further comprises DHA.
 6. The method according to claim 1, wherein the composition comprises 7.5 to 12.5 energy % protein; 40 to 55 energy % carbohydrates; and 35 to 50 energy % fat, wherein said protein comprises hydrolyzed milk protein, vegetable protein, amino acids, or combinations thereof.
 7. The method according to claim 1, wherein the composition comprises a caloric content of 0.6 to 0.8 kcal/ml; an osmolality of 50 to 500 mOsm/kg; and a viscosity below 50 mPas.
 8. The method according to claim 1, wherein: (a) the content of long chain polyunsaturated fatty acid is below 3 wt. % of the total fat content; (b) the content of omega-3 long chain polyunsaturated fatty acid is below 1 wt. % of the total fat content; and (c) the content of omega-6 long chain polyunsaturated fatty acid is below 2 wt. % of the total fat content.
 9. The method according to claim 1, wherein the composition further comprises between 5 and 100 mg nucleosides and/or nucleotides per 100 gram dry formula.
 10. The method according to claim 1, wherein the composition is an infant formula.
 11. A method for stimulating intestinal barrier integrity comprising administering to a subject in need thereof, a nutritional composition comprising: (a) eicosapentaenoic acid (EPA) and arachidonic acid (ARA), wherein the content of long chain polyunsaturated fatty acid with 20 and 22 carbon atoms does not exceed 15 wt. % of the total fat content; and (b) at least two distinct oligosaccharides with a degree of polymerisation (DP) between 2 and 60 comprising transgalactooligosaccharide (TOS) and inulin.
 12. The method according to claim 11, wherein at least 10 wt. % of the oligosaccharides has a degree of polymerisation (DP) between 2 to 5 and at least 5 wt. % of the oligosaccharides has a DP between 10 and
 60. 13. The method according to claim 11, wherein the composition further comprises an acidic oligosaccharide with a DP between 2 and
 60. 14. The method according to claim 13, wherein the acidic oligosaccharide is an uronic acid polymer with a DP between 2 and
 6. 15. The method according to claim 11, wherein the composition further comprises DHA.
 16. The method according to claim 11, wherein the composition comprises 7.5 to 12.5 energy % protein; 40 to 55 energy % carbohydrates; and 35 to 50 energy % fat, wherein said protein comprises hydrolyzed milk protein, vegetable protein, amino acids, or combinations thereof.
 17. The method according to claim 11, wherein the composition comprises a caloric content of 0.6 to 0.8 kcal/ml; an osmolality of 50 to 500 mOsm/kg; and a viscosity below 50 mPas.
 18. The method according to claim 11, wherein: (a) the content of long chain polyunsaturated fatty acid is below 3 wt. % of the total fat content; (b) the content of omega-3 long chain polyunsaturated fatty acid is below 1 wt. % of the total fat content; and (c) the content of omega-6 long chain polyunsaturated fatty acid is below 2 wt. % of the total fat content.
 19. The method according to claim 11, wherein the composition further comprises between 5 and 100 mg nucleosides and/or nucleotides per 100 gram dry formula.
 20. The method according to claim 11, wherein the composition is an infant formula. 